The present invention relates generally to lysosomal storage disorders and to diagnostic agents for their detection in humans and other animals. More particularly, the present invention is directed to the uses of the LSD markers Lamp-1, Lamp-2. Limp-II, 4-sulphatase, acid phosphatase (ACP), xcex2-hexosaminidase or xcex1-mannosidase, amongst others as diagnostic agents for the detection of many lysosomal storage disorders.
Bibliographic details of the publications referred to in this specification by author are collected at the end of the description.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.
Lysosomal storage disorders (LSD) represent a group of 39 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins which are involved in lysosomal biogenesis. The importance of these disorders to health care becomes obvious when the group incidence rate for LSD (1:5,000 births) is compared with well known and intensively studied genetic disorders, for which newborn screening is currently performed, such as phenylketonuria (1:14,000) and cystic fibrosis (1:2,500). LSD generally affect young children and have a devastating impact on the child and the family involved. Affected individuals can present with a wide range of clinical symptoms depending upon the specific disorder and the particular genotype involved. Central nervous system dysfunction, from behavioural problems to severe mental retardation, is characteristic of many LSD. In the mucopolysaccharidoses, other symptoms may include skeletal abnormalities, organomegaly, corneal clouding and dysmorphic features (Neufeld and Meunzer, 1995). In severe cases, the child requires constant medical management of the disorder but dies before adolescence.
Except for those cases with a family history of the disease, pre-symptomatic detection of LSD can only be achieved by newborn screening. Currently, even after the presentation of clinical symptoms, the diagnosis of a LSD is a complex process involving a range of assays performed on urine, blood and in some disorders, skin fibroblasts. These assays are time consuming, expensive and invasive, making them unsuitable for newborn screening applications. In order to justify the screening of the entire neonatal population for a given disorder or group of disorders there are a number of criteria which need to be satisfied, these criteria can be summarised as two broad considerations. Firstly, does neonatal diagnosis provide clear cut benefits to the neonate and family? Secondly, are these benefits reasonably balanced by the total cost of screening?
In recent years, treatment of some LSD has become possible. Cystinosis is treated with cysteamine (Gahl et al., 1987; Markello et al., 1993), a number of LSD including mucopolysaccharidosis (MPS) I and MPS VI have been responsive to bone marrow transplants (Hoogerbrugge et al, 1995; Hopwood et al, 1993) and Gaucher disease is currently being treated by enzyme replacement therapy which, like bone marrow transplantation, is theoretically applicable to a wide range of LSD. Recombinant enzymes deficient in many of LSD have been characterised and there are now numerous animal models which are being used to evaluate enzyme replacement and gene therapies for these disorders. Animal models currently in use include dog models for fucosidosis (Taylor et al., 1989) and MPS VII (Haskins et al. 1992), cat models for MPS I, and VI (Crawley et al., 1996; Haskins et al, 1992), goat models of xcex2-mannosidosis (Jones and Kennedy, 1993) and MPS IIID (Thompson et al., 1992) and mouse models for MPS VII (Sands et al., 1994), galactosialidosis (Zhou et al., 1995) and Niemann-Pick disease (Otterbach and Stoffel, 1995). It is probable that within the next 5 to 10 years effective therapies will be available for many of the LSD.
The effectiveness of these therapies, particularly for those LSD involving central nervous system and bone pathologies, will rely heavily upon the early diagnosis and treatment of the disorder, before the onset of irreversible pathology. Animal studies involving bone marrow transplantation in a fucosidosis dog model, which relates predominantly to central nervous system pathology (Taylor et al., 1989) and enzyme replacement therapy studies in an MPS VI cat model (predominantly bone pathology) (Crawley et al., 1996; Crawley et al., 1997) have shown a clear correlation between the age when treatment was commenced and efficacy and that enzyme replacement therapy is effective for the prevention of bone pathology.
A further consideration, critical to bone marrow transplant therapy, is that early diagnosis of the LSD will allow clinicians to take advantage of the window of opportunity presented by the naturally suppressed immune system of the neonate to maximise the chances of a successful engraftment.
Early detection of these disorders has the added advantage of permitting genetic counselling of the parents, with the option of prenatal diagnosis in subsequent pregnancies, and management of the affected child. Accurate techniques for monitoring progress of the treatment regimes are also required.
One common feature of these LSDs is the accumulation and storage of material normally degraded within the lysosome and transported across the lysosomal membrane. It is generally recognised that this results in an increase in the number and size of lysosomes within the cell from approximately 1% to as much as 50% of total cellular volume. However, although the formation of lysosomal storage vacuoles within affected cells is well-known, the process by which lysosomal biogenesis occurs, in particular the nature and role of genes and enzymes which are involved in the process, is poorly understood.
In work leading up the present invention, the inventors sought to identify proteins nucleic acid molecules, oligosaccharides, gangliosides and processes involved in lysosome biogenesis, which are capable of functioning as markers of lysosome storage disorders (hereinafter referred to as xe2x80x9cLSD markersxe2x80x9d). The LSD markers identified by the inventors have provided for the development of a wide range of diagnostic and therapeutic reagents for the treatment of LSDs in humans and other animals, including the development of procedures to facilitate the presymptomatic detection of all LSDs in a single assay.
Accordingly, one aspect of the present invention provides a diagnostic method of detecting a lysosomal storage disorder (LSD), monitoring the progress of an LSD or the efficacy of treatment of an LSD in a human or other animal patient comprising assaying the level of expression of an LSD marker as defined herein in a biological sample derived from said patient.
As used herein, the term xe2x80x9cLSD markerxe2x80x9d or similar term shall be taken to refer to an enzyme, protein, polypeptide or other biomolecule or a homologue, analogue or variant thereof derived from the lysosome of a human or other animal, the presence or level of expression of which is associated with the occurrence, development or onset of at least one LSD in said animal. An LSD marker is usually expressed in a cell derived from a patient having an LSD at a level which is different from that observed for a normal individual.
The present invention extends to the assay of an LSD marker for the diagnosis of a wide range of LSDs selected from, but not limited to the list comprising Pompe disease, Salla disease, Gaucher disease, mucopolysaccharidoses (MPS) including MPS I, MPS II, MPS IIIIA, MPS IIIB, MPS IIIIC, MPS IVA, and MPS VI, I-cell disease including ML II/III, Tay-Sach""s disease. Fabry""s disease, metachromatic leukodystrophy (MLD), Niemann-Pick disease and multiple sulphatase deficiency, amongst others.
Those skilled in the art will be aware that a marker may also be used to diagnose a genetic predisposition toward the disease which the marker is used to detect. The present invention therefore extends to the assay of an LSD marker for determining the genetic predisposition of a patient to one or more or the LSD discussed supra.
An LSD marker according to the present invention may be any lysosomal enzyme, protein, polypeptide or other biomolecule which is up-regulated as a result of the lysosomal proliferation which is characteristic of an LSD or at least accumulates at an increased rate in the lysosomes of patients suffering from an LSD. Those skilled in the relevant art will be aware that the most suitable LSD markers for the present purpose are those enzymes, proteins, polypeptides or other biomolecules which are expressed at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold and even more preferably at least 20-fold higher in the cells of LSD-affected patients than in non-affected patients.
The present invention extends to the use of any one or more of Lamp-1, Lamp-2, Limp-II, mannose-6-phosphate receptors, 4-sulphatase, acid phosphatase (ACP), xcex2-hexosaminidase, or xcex1-mannosidase, amongst others as an LSD marker.
The invention further extends to the use of the foregoing LSD markers in the manufacture of a composition or medicament for the diagnosis and/or treatment of an LSD in a human or animal subject.
In a particularly preferred embodiment of the present invention, said LSD marker is the lysosomal Lamp-1 protein. As described in the Examples herein, the inventors have found that the level of Lamp-1, protein is elevated in a wide range of patients suffering from LSDs, when compared to the level of Lamp-1 expression in normal individuals. For example, the level of Lamp-1 protein is 3- to 6-fold higher in the plasma obtained from a patient suffering MPS I compared to a non-affected individual.
In one embodiment of the invention, the level of expression of said LSD marker is assayed by measuring the level of enzyme activity of said LSD marker. Several methods are available for the assay of particular enzymes derived from biological samples. Those skilled in the art will be aware that an assay method will vary depending upon the nature of the LSD marker in question, including its substrate preference and co-factor requirement and the tissue or organ from which it was derived. Assay methods for the lysosomal enzymes ACP, xcex2-hexosaminidase, xcex1-L-iduronidase and xcex1-mannosidase are incorporated herein by way of exemplification only.
In an alternative embodiment, wherein said LSD marker is either a protein or polypeptide or other stored substrate, the level of expression of said LSD marker may be assayed by an immunoassay. Those skilled in the art are aware that, in its broadest context, an xe2x80x9cimmunoassayxe2x80x9d comprises incubating a test sample with one or more immunointeractive molecules specific for said LSD marker, for example an antibody, for a time and under conditions sufficient for binding thereto and detecting said binding. Altered levels of the LSD marker, in particular elevated levels of the LSD marker Lamp-1, compared to the levels detected in non-affected patients, may indicate an LSD.
Conditions for incubating an antibody with a test sample vary, depending upon the format employed in the assay, the detection methods employed and the type and nature of the antibody molecule used in the assay. Those skilled in the art will recognise that any one of the commonly available immunological assay formats, for example radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), diffusion-based Ouchterlony, rocket gel immunoelectrophoresis or in situ immunoassays can be readily adapted to the present purpose. Examples of such assay formats can be found in Chard (1986), Bullock (1982, 1983, 1984) or Tijssen (1985). Generally, the assay format will be selected to provide the highest sensitivity of detection for the test sample.
Immunoassays are useful in the quantification of an LSD marker in a test sample, particularly test samples derived from blood samples or isolated cells, in particular to determine whether the level of said LSD marker is elevated compared to normal levels detectable in non-affected individuals. As a consequence, such an immunoassay is of particular use in determining whether a patient may have a lysosomal storage disorder. The invention described herein extends to all such uses of immunointeractive molecules and diagnostic assays which require said immunoassays for their performance.
A wide range of immunoassay techniques may be used, such as those described in U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. By way of example only, an antibody raised against the Lamp-1 protein is immobilised onto a solid substrate to form a first complex and a biological test sample from a patient is brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-Lamp-1 secondary complex, a second Lamp-1 antibody labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing sufficient time for the formation of a tertiary complex of antibody-Lamp-1-labelled antibody. Any unreacted material is washed away, and the presence of the tertiary complex is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal or may be quantitated by comparison with a control sample containing known amounts of hapten. Variations of this assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be readily apparent. The antibodies used above may be monoclonal or polyclonal.
The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.
By xe2x80x9creporter moleculexe2x80x9d, as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecule in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, xcex2-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product.
Alternatively, fluorescent compounds, such as fluorescein, Eu3+ or other lanthanide metals, and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. It will be readily apparent to the skilled technician how to vary the procedure to suit the required purpose.
The immunologically-interactive molecule, in particular an antibody molecule, is also useful in purifying an LSD marker protein or in the manufacture of a compound or medicament for the diagnosis and/or treatment of an LSD in a human or animal subject. Methods for the affinity purification of proteins using antibodies are well-known to those skilled in the art.
In a particularly preferred embodiment, the immunoassay employed according to the invention is an ELISA. Antibodies labelled with Eu3+ or other lanthanide metals may also be useful as detection molecules in immunoassays based on the time delayed fluorescence, observed with these compounds.
The immunoassay test samples of the present invention may be derived from any organ, tissue or other biological sample comprising lysosomes. Accordingly, the diagnostic assay of the present invention may be carried out using test samples derived from a human or other animal of any developmental stage including a foetus, embryo, neonate or adult animal, provided that the sample contains a sufficient level of said LSD marker to be detected using a known assay format. Suitable test samples include, but are not limited to crude or partially-purified extracts from cells such as fibroblasts, cultured cell lines, urine, blood and blood-derived products such as serum or plasma, amongst others.
The test sample used in the above-described method will vary based upon the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts of cells are well-known in the art and can be readily adapted in order to obtain a sample which is suitable for the assay format selected.
In a particularly preferred embodiment of the present invention, the test sample is a blood sample or plasma, for example a blood-spot taken from a Guthrie card.
The diagnostic methods described supra are useful in antenatal screening for LSD. The invention is also of particular utility in the screening of neonates up to 7 days of age using dried blot spots collected from infants. However, the present invention extends to the use of any assay format or test sample to detect an LSD marker.
A second aspect of the present invention provides a biomolecule to facilitate the detection of a lysosomal storage disorder in a human or other animal, wherein said biomolecule is capable of binding to an LSD marker as defined herein when used in an assay to determine the level of expression of said LSD marker in a biological test sample derived from said human or other animal.
According to this aspect of the invention, said biomolecule may be an enzyme substrate molecule, a co-factor, an immunologically interactive molecule such as an antibody molecule.
In one embodiment, the biomolecule according to this aspect of the invention is an immunologically interactive molecule.
The term xe2x80x9cimmunologically interactive moleculexe2x80x9d as used herein shall be taken to refer to a polyclonal or monoclonal antibody or a functional derivative thereof, for example a Fab, SCAB (single-chain antibody) or an antibody conjugated to an enzyme, radioactive, paramagnetic or fluorescent tag, the only requirement being that said immunologically interactive molecule is capable of binding to an LSD marker or a derivative, part, fragment, analogue or homologue thereof.
Preferably, the immunologically interactive molecule is in the form of an antibody such as a polyclonal or monoclonal antibody. The present invention extends to immunologically interactive fragments, parts, derivatives, homologues or analogues of these antibodies. Such antibodies may be in an isolated or purified form comprising at least 25% (w/w), more preferably at least 50% (w/w), even more preferably at least 60-75% (w/w) and even still more preferably at least 80-95% (w/w) of immunoglobulin on a protein basis. Alternatively, the antibodies may be present in the form of isolated hybridoma, culture supernatant, tissue extract, serum or whole blood or ascites fluid.
Conventional methods can be used to prepare the immunologically interactive molecules. By using a polypeptide comprising all or a fragment of an LSD marker as defined herein, polyclonal antisera or monoclonal antibodies can be made using standard methods. For example, any mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of an antigen comprising an LSD marker to elicit an antibody response in the mammal. Techniques for conferring immunogenicity on an antigen include conjugation to carriers or other techniques well known in the art. For example, the antigen can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired IgG molecules corresponding to the polyclonal antibodies may be isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985), and screening of combinatorial antibody libraries (Huse et al., 1989). Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the antigen and monoclonal antibodies isolated.
As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the polypeptides of the invention must be determined empirically. Factors to be considered include the immunogenicity of the native antigen, whether or not the antigen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the composition, i.e. intravenous, intramuscular, subcutaneous, etc. and the number of immunizing doses to be administered. Such factors are known in the art and it is well within the skill of immunologists to make such determinations without undue experimentation.
The term xe2x80x9cantibodyxe2x80x9d as used herein, is intended to include fragments thereof which are also specifically reactive with a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of an LSD marker according to the embodiments described herein, in particular the Lamp-1 and 4-sulphatase. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(abxe2x80x2)2 fragments can be generated by treating antibody with pepsin. The resulting F(abxe2x80x2)2 fragment can be treated to reduce disulfide bridges to produce Fabxe2x80x2 fragments.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of a polypeptide which comprises, mimics, or cross-reacts with a B cell or T cell epitope of an LSD marker as hereinbefore defined.
The polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect and/or quantify an LSD marker or a derivative, homologue or analogue thereof in various biological materials, for example they can be used in an ELISA, radioimmunoassay or histochemical tests. Thus, the antibodies can be used to test for binding to an LSD marker or a derivative, homologue or analogue thereof in a sample or to determine B cell or T cell epitopes of an LSD marker.
Accordingly, in another embodiment of the present invention, the above-described antibodies are detectably labeled to facilitate their use in immunoassays conducted either in vitro, in vivo or in situ. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels such as biotin, avidin and the like, enzyme labels such as horse radish peroxidase, alkaline phosphatase and the like, fluorescent labels such as FITC, rhodamine and the like or Eu3+, using paramagnetic atoms, amongst others. Procedures for accomplishing such labeling are well-known in the art.
Preferably, the antibody is raised against a protein or polypeptide. However, in a particularly preferred embodiment, the present invention provides antibodies which are capable of recognising either the Lamp-1, Lamp-2, Limp-II or 4-sulphatase protein as described herein.
A further aspect of the invention contemplates a diagnostic kit for determining the level of expression of an LSD marker in a biological sample derived from a human or other animal suspected of having a lysosome storage disorder.
In a preferred embodiment of this aspect of the invention, said kit comprises in a first compartment several first containers adapted to contain an LSD marker enzyme, protein, polypeptide or a derivative, homologue or analogue thereof in recombinant or synthetic form and optionally adsorbed thereto, and several second containers adapted to contain an antibody which recognises said LSD marker or a B cell or T cell epitope thereof, wherein said antibody is optionally labelled with a reporter molecule capable of producing a detectable signal as hereinbefore described. There are also provided several third containers which contain a second antibody which recognises the first antibody and is optionally conjugated to a reporter molecule. If the reporter molecule is an enzyme, then several fourth containers are provided which contain a substrate molecule for said enzyme to facilitate detection of the enzyme:antibody:LSD marker complex or alternatively, the enzyme:antibody:antibody:LSD marker complex where a second antibody has been used. The reporter molecule used in this kit may also be a radio-isotope, a fluorescent molecule, or bioluminescent molecule, amongst others.
Optionally, the kit may further be contained in a package which comprises microtitre wells in one section, in which reactions may be performed. Accordingly, in one embodiment, the microtitre wells may be the equivalent of the first compartment hereinbefore described and contain the LSD marker or a derivative, homologue or analogue thereof, adsorbed thereto.
Optionally, the first, second, third and fourth containers of said kit may further be colour-coded for ease-of use.
In an exemplified use of the subject kit, the contents of the first container may be bound to a microtitre well contained in the package, if not provided in a format where said contents are already adsorbed to said microtitre well, and a biological sample to be tested is added and incubated for a time and under conditions sufficient for an antigen-antibody complex to form in said microtitre well. Following a washing step to remove unbound antibodies and other unbound protein, the contents of the third container are added to the antigen-antibody complex contained in the microtitre well and the reaction allowed to proceed for a time, and under conditions sufficient to allow the formation of the tertiary antigen-antibody-antibody complex. A control reaction may be performed in which the contents of the second container are added to the contents of the first container for a time and under conditions suitable for the formation of an antigen-antibody complex. If the antibody of the second container is not labelled with a reporter molecule, then the contents of the third container may be added for a time and under conditions suitable for the formation of a tertiary antigen-antibody-antibody complex to form. The tertiary antigen-antibody-antibody complexes of the control reaction and the test sample are then subjected to a detecting means. Alternatively, if the contents of the second container are labelled with a reporter molecule the antigen-antibody complex of the control reaction may be subjected directly to a detecting means. The means of detection of a secondary antigen-antibody or a tertiary antigen-antibody-antibody complex are numerous, as hereinbefore described and will be known to those skilled in the art. Where said means is an enzyme reaction, the contents of the fourth container are added to said secondary or tertiary complex thus formed for a time and under conditions suitable to enable the enzyme reaction to occur.
In analysing the results obtained using the subject kit, the amount of LSD marker contained in the control reaction is predetermined to provide a result which is consistent with the result obtained for a normal non-affected patient and therefore the control reaction provides a basis for comparison with the test sample. A signal obtained for the test sample which is higher than that of the control indicates a higher level of the subject LSD marker being tested. Such a result may indicate that the patient is suffering from a lysosomal storage disorder.
The present invention further extends to any kit comprising a biomolecule which is capable of detecting a lysosomal storage disorder in a human or other animal, wherein said kit is in a form which is suitable for an assay to detect expression of an LSD marker as hereinbefore defined. The present invention also extends to kits comprising multiple of said biomolecules to facilitate the detection of more than one LSD marker.